Isolation of Bioluminescent Bacteria and Their Application in Toxicity Testing of Chromium in Water

Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 23-32

ISSN: 2319-7706 Volume 4 Number 10 (2015) pp. 23-32 http://www.ijcmas.com

Original Research Article Isolation of Bioluminescent Bacteria and Their Application in Toxicity Testing of Chromium in Water

Varsha Shivaji Kulkarni* and Bhagyashree Shashikant Kulkarni

Department of Microbiology, Sinhgad College of Science, Ambegaon (Bk), Pune-411046, India *Corresponding author

A B S T R A C T

Nature has its own beauty and phenomenon called bioluminescence proves it. Marine fishes like Japanese Threadfin brin (Nemipterus japonicas), Indian Mackerel (Restrelliger kanagurta) and Anchovy (Engraulis japonicas) were chosen as source of bioluminescent bacteria. Scales, gills and eye portion along with gut region of Indian Mackerel were suspended overnight in sterile sea salt saline and from this loopful suspension were streak inoculated on sterile Photobacterium agar, LA, Sea water agar and Boss Agar. By subsequent subculturing two luminescent isolates K e y w o r d s named as C1, C2 were obtaining on LA plate from the gut portion of the Indian Mackerel. They were used for further study. Morphological and biochemical Biolumine- characterization along with observation of PHB granules under light microscope scence, suggests that the isolates may belong to Genus Vibrio. To reconfirm these results the Luciferase, isolates were inoculated on TCBS agar which is the highly specific media for the Auto- Vibrio identification and confirmation; which confirmed that the isolate C1 may be induction, late sucrose fermenter Vibrio species. This isolate showed green coloured colonies Pollutant after 36hrs of incubation. The TSI slant examination stated that the organisms were chromium, Gram negative enteric pathogen that ferments glucose, lactose and sucrose by acid Toxicity production without accumulation of gas. After examination of growth pattern of both testing isolates; C1 and C2 were used for the checking toxicity of the chemical pollutant cultivation chromium in water. According to the United States Environmental Protection Agency for primary drinking water the standard Maximum Contaminant Level is 0.1 mg/L for total chromium. Standard protocols were used to determine total chromium level in water. Performance of the TVC of the both isolates lead to the results that the pollutant chromium inhibits the growth of bioluminescent bacteria and also affects the luminescence intensity. This whole study provides eco friendly and cost effective examination of pollutant chromium. The method can also be used to measure other chemicals, metal salts like mercuric chloride, zinc chloride and lead acetate etc.

Introduction

Bioluminescence a Greek word ( Bios for This type of chemiluminescent phenomenon living and Latin word lumen for light ) have been seen in many types of organisms which literally means a living light. (bacteria, fungi, fishes, squid,

Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 23-32 dinoflagellates) and have always gained an bacteria and algae gaining the attention for a attention of scientists because of their toxicity evaluation as a model organisms beauty, physiology, biochemistry, genetics (Roda et al., 2004; Boynton, 2009). As well and ease of detection (Lee 2008; known; general metabolism, operon Vedprakash et al., 2014). In late 19th century expression, bacterial growth, cultural Raphael Dubois experimentally extracted condition affects the luminescence activity; the two key components, enzyme from which one can articulate that the luciferase and luciferine of bioluminescent organisms are very much bioluminescence reaction which are able to sensitive to any change (e.g. physical, generate light (Makemson, 1986; Lee, chemical, radiological, etc.) in environment. 2008). Many bacteria regulate their set of Bioluminescent bacterial tests have been lux genes by the mechanism of quorum successfully used previously in determining sensing or autoinduction in which the toxicity of aquatic samples, sediments, autoinducers (AI) evoke the characteristic and soils (Lapota et al., 1998; Kurvet et al., response from the cell (Rees et al., 1998; 2005). Nyholm et al., 2004; Walker et al., 2006). In bioluminescence after achieving specific Materials and Methods

Anchovy (local name: Mandeli); Indian threshold value (conc. of AI 10 cell mL ¹ in Mackerel (local name: Bangda); and Threadfin brin (local name: Raja Rani) these three fresh marine water fishes were selected culture media) it triggers the synthesis of as a source of bioluminescent bacteria and enzyme luciferase and other enzymes were obtained from fish market near Alpna involved in luminescence (Cao and Meighen Talkies, Camp, Pune. They were kept on ice and soon after brought to the laboratory for further processes. 1989; Rees et al., 1998 Walker et al., 2006; Following fish parts were used as a source for luminescent bacteria (Table 1) (Walker et al., 2006, web links).

Berleman, 2009 . This sensing (AI level) Table.1 Fish parts used for luminescent bacteria help to ensure that the luminescence Eye Gill Scales Fin Gut production is enough high to cause an portion region impact in environment and making the Anchovy - Used Used - - process cost effective (Nealson et al., 1970; Mackerel - Used Used - Used Visick et al., 2000). As the production of the Fim Used Used Used Dorsal - luminose enzyme increases ultimately light bream fin generation also increases (Nealson et al., 1970; Cao and Meighen, 1989; Berleman, These different parts were suspended in 2009). sterile sea saline that is 3.5g of sea salt in 100ml distilled water for 24hrs and from this Due to the issues raised by animal rights and loopful suspension was streaked for human rights, the use of microbes like isolation (Web link) on sterile

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Photobacterium agar (PBA) casein enzyme selective for Vibrios and differential due to hydrolysate 5g/L, yeast extract 2g/L, presence of sucrose and the dyes. TCBS ammonium chloride 0.3g/L, magnesium agar (proteose peptone 10g/L, yeast extract sulfate 0.3g/L, ferric chloride 0.01g/L, 5g/L, sodium thiosuphate 10g/L, sodium calcium chloride 1g/L, potassium phosphate citrate 10g/L, oxagall 8g/L, sucrose 20g/L, (monobasic) 3g/L, sodium glycerophosphate NaCl 10g/L, ferric citrate 1g/L, 23.5g/L, sodium chloride 30g/L, agar 15g/L bromothymol blue 0.40g/L, thymol blue (web link), sea water agar Part A: yeast 0.40g/L, agar 15g/L, pH 8.6±0.2 at 25 C, extract 5g/L, peptic digests of animal tissue formula adjusted, standardized) is highly 5g/L, beef extract 3g/L, agar 15g/L, Part B: selective, meets the nutritional requirements sodium chloride 24g/L, potassium chloride of Vibrio spp., and allows Vibrios to 0.7g/L, magnesium chloride. 6H O 5.3g/L, compete with intestinal flora (Engebrech et magnesium sulfate. 7H O.7g/L, calcium al., 1983; Musa et al., 2008; Ransanga et al., chloride0.1g/L, pH 7.3 +/ 0.2 (web link), 2012; Kannahi and Shivshankari, 2014) Luminus agar (LA) sodium chloride 10g/L, TCBS Agar is recommended by APHA yeast extract 5g/L, peptone10g/L, (American Public Health Association) for agar15g/L, pH 7.3 (web link) and the selective isolation of V. cholerae and V. bioluminescent agar (Boss agar) sodium parahaemolyticus. Enrichment in alkaline chloride30g/L, glycerol1g/L, peptone10g/L, peptone water (M618), followed by isolation meat extract3g/L, pH 7.3 plates (web link). on TCBS Agar is routinely used for isolation Plates were incubated at RT for 48 72hrs of V. cholerae. Vibrios are natural and observed for bioluminescence (web inhabitants of sea water (Kumar, 2010). It is link). possible that a few sucrose-positive Proteus strains can grow to form yellow colonies. Identification of isolated bioluminescent Vibrio parahaemolyticus is a sucrose non- species fermenting organism and produces blue- green colonies, as does Vibrio vulnificus Two isolates were obtained by subsequent (Engebrech et al., 1983; Musa et al., 2008; sub-culturing on sterile LA plates. The Orengo et al., 2010; Ransanga et al., 2012; isolated were named as C1 and C2. After Kannahi and Shivshankari, 2014). morphological colony characterization, Gram staining, motility and Poly- -hydroxy- Triple sugar iron agar (TSI) butyrate staining (by Sudan Black B method) were performed (Bergey s manual TSI is recommended in the identification of 9th edition, web link). Bergey s Manual of gram-negative enteric pathogens in routine determinative Bacteriology 9th edition was examination of faeces. This medium referred and accordingly biochemical tests indicates the ability of an organism to like IMViC, amylase, oxidase, nitrate ferment lactose, sucrose and glucose with reduction were performed and fermentation formation of acid and gas and also its ability of sugars were also checked with 12 to produce hydrogen sulfide gas (Musa et different sugars (Table 2). al., 2008; Kumar, 2010; Ransanga et al., 2012; Kannahi and Shivshankari, 2014) Growth on Thiosulfate Citrate Bile (Table 3 and 4). Sucrose Agar (TCBS) Growth curve TCBS Agar is both differential and highly

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Loopful of 24 hr old culture of isolate C1 Orange Indicator solution, Conc. and isolate C2 were inoculated in 50 ml Ammonium hydroxide, Potassium Sterile Luminous Broth(LB) and were kept permanganate solution: Dissolve 4 g overnight till absorbance reached 0.1 at 600 KMnO in 100ml water, Diphenylcarbazide nm. After 0.1 absorbance was reached, solution: Dissolve 250 mg in 50ml acetone growth curve experiment was performed by and Store in brown bottle (APHA, 2012). taking absorbance at 600nm at 30 minutes Protocol: (APHA, 2012; Tchobanoglous et interval time period (Eberhard, 1972; al., 2004). Pipette a portion of digested Engebrech et al., 1983; Cao and Meighen, sample into 125 ml conical flasks, Add few 1989; Kumar, 2010; Gokhale et al., 2012) drops of methyl orange indicator and then (Graph 1 and 2). add conc. Ammonium Hydroxide until solution just begins to turn yellow, Add Application of bioluminescent bacteria in 1+11 ml of sulphuric acid until it turns toxicity testing of chromium acidic+1ml sulphuric acid in excess, Adjust the volume to 40ml, Add porcelain pieces The bacterial bioluminescence test (BBT) is and boil, Add 2 drops of potassium metabolic inhibition test that uses a permanganate solution to give a dark red standardized suspension of luminescent colour, if it fades add more potassium bacteria as test organisms under permanganate, Boil for 2mins, Add 1ml standardized conditions. This test method sodium azide solution, If red colour doesn t provides a rapid, reliable and convenient fade completely after boiling for means of determining the toxicity of waste approximately 30 seconds, add another 1ml material. We have performed this test for sodium azide solution and continue boiling checking out the chromium (heavy metal) for 1 min, after color has faded completely, toxicity on one of our bioluminescent then cool, Add 0.25 ml ortho-phosphoric bacterium. 0.1 mg of potassium dichromate acid and using 0.2N sulphuric acid adjust pH was added in 100ml distilled water (APHA, to 1 for yellow colour development and take 2012, Tchobanoglous et al., 2004). O.D. at 540 nm. A 24 hour old culture was Following protocol was used to convert total inoculated in sterile Luminous Broth to chromium to the hexavalent state chromium adjust the O.D of 0.1 at 600nm absorbance. by oxidation with potassium permanganate. C2 was used for this testing. 4ml of such The oxidation process may not provide total absorbance adjusted broth culture was conversion of all chromium species to Cr . exposed 1ml of different metal salt solution For total chromium determination, acid- that is potassium dichromate (K Cr O ) at digestion of the sample was done. The different concentration up to 60 minutes. hexavalent chromium is determined Samples were withdrawn at different time colorimeterically by reaction with intervals of 10, 20, 30, 40, 50, 60 minutes diphenycarbazide in acid solution. A red- and serially diluted up to 10 . Dilutions violet colures complex of unknown 10 ³and 10 were plated out on sterile LA composition is produced. The reaction is plates. Sample of unexposed culture was very sensitive, the molar absorbstivity based plated out as control (Eberhard, 1972; on chromium being about 40,000 L /g/cm Engebrech et al., 1983, Cao and Meighen, at540nm (APHA, 2012; Tchobanoglous et 1989; Kumar, 2010, Gokhale et al., 2012; al., 2004). APHA, 2012; Tchobanoglous et al., 2004). Chemicals Required: Conc. Nitric Acid, Conc. Ortho-phosphoric acid, Methyl Results and Discussion

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Orengo et al., 2010; Ransanga et al., 2012; Observing plates in dark revealed that the Kannahi and Shivshankari, 2014) (Table 2). colonies obtain from the gills and scales of To reconfirm the present results both of the Fim Brim inoculums and gut of Indian isolates were streak inoculated on TCBS Mackerel inoculums were glowing agar which is specific for Vibrio beautifully also in presence of other identification (Musa et al., 2008; Kumar, bacterial population. After subsequent sub- 2010, Ransanga et al., 2012). Our finding culturing on LA it had been observed that indicates that the isolate C2 may be late the colonies were obtain from the gut flora sucrose fermenters as it showed green of Indian Mackerel were glowing with more coloured colonies after 36hrs which was intensity than the colonies obtain from gills showing yellow colonies after 24hrs of and scales of Fim Brim. And because of this incubation while C1 showed green colonies these colonies were chosen for the after 24hrs of incubation. Hydrogen sulfide experimental purpose (Engebrech et al., gas production was absent for both C1 and 1983; Musa et al., 2008; Orengo et al., C2 (Musa et al., 2008; Ransanga et al., 2010; Ransanga et al., 2012; Kannahi and 2012). These results confirm that both the Shivshankari, 2014; web link). Two isolates were from Genus Vibrio (Engebrech different bioluminescent isolates were et al., 1983; Musa et al., 2008; Orengo et observed on the LA plates and were al., 2010; Kumar, 2010, Ransanga et al., examine for further studies (Photo 1). 2012; Kannahi and Shishankari, 2014). Microscopic examination of these two Inoculation of C1 and C2 on TSI slants isolates states that the circular, creamy white identify that organisms were Gram negative colonies having convex elevation with enteric pathogens (Musa et al., 2008; smooth (C1) and Mucoid (C2) consistency Ransanga et al., 2012) C1 fermented the were Gram negative motile short rods lactose or sucrose or both the sugars in (Bergey s Manual, 9th edition) Both of the absence of H S in acidic condition while C2 isolates C1 and C2 found to be poly- - fermented glucose by producing acid in hyrdoxy butarate granules non producers; as absence of gas (Engebrech et al., 1983; no accumulation of PHB were seen in the Musa et al., 2008; Orengo et al., 2010; cells after staining by Sudan Black B Kumar, 2010, Ransanga et al., 2012; method (Bergey s Manual, 9th edition; Kannahi and shishankari, 2014) (Table 3 Ransanga et al., 2012). By referring and 4). The important growth curve Bergey s manual of determinative experiment of both the isolates indicates that bacteriology (9th edition), biochemical test there was rapid increase in absorbance were performed on both the isolates. Both of showed by C1 and C2 isolates were showing the isolates were able to produce enzyme steady increase in cell number. This gelatinase, catalase and cytocrome oxidase information gave an idea regarding when and showed negative results for IMViC and cells are going to be in log phase. In log enzyme amylase production. All 12 chosen phase bioluminescent activity is more sugars were fermented by C1 and C2 with prominent (Eberhard, 1972; Cao and acid production in absence of gas (Bergey s Meighen, 1989; Gokhale et al., 2012) and all Manual, 9th edition; Musa et al., 2008; because of this reason this experiment was Ransanga et al., 2012). These results the key for the whole toxicity experiment indicates that the both isolates belong to the (Eberhard, 1972; Engebrech et al., 1983; genus Vibrio (Bergey s Manual, 9th edition; Cao and Meighen, 1989; Kumar, 2010; Engebrech et al., 1983; Musa et al., 2008; Gokhale et al., 2012) (Graph 1 and 2).

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Heavy metal toxicity testing 10 ³ and 10 dilution. Comparison with These bioluminescent bacteria were used to check toxicity of heavy metal chromium. Chromium may exist in water supplies in control plates which was free from both the hexavalent and trivalent form chromium ions showed presence of although trivalent form rarely occurs in 396CFU/ml ×10 but it was found to be potable water. The U.S. EPA (United States gradually decreasing as found in table 5. Environmental Protection Agency) primary These results conclude that the presence of drinking water standard MCL (Maximum chromium affect the bioluminescent Contaminant Levels) is 0.1 mg/L for total bacterial growth. So the use of this chromium (APHA, 2012; Tchobanoglous, bioluminescent bacteria for the toxicity 2004). So serially diluted sample was testing was successfully accomplished with checked for the total viable count between easy detection (APHA, 2012; Tchobanoglous, 2004).

Table.2 Characteristic and biochemical test results

Isolate C-1 C-2 Morphology Gram character -/S -/S Motility + + Consistency Smooth Mucoid PHB Production - - Luminescence +/blue colour +/green colour Fermentation of sugar Arabinose A A Fructose A A Galactose A A Glucose A A Lactose A A Maltose A A Manitol A A Mannose A A Rhamnose A A Ribose A A Sucrose A A Xylose A A Biochemical Tests Indole test - - Methyl Red test - - Voges-Prosker test - - Citrate - - Nitrate Reduction test - - Catalase test + + Amylase test - -

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Gelatinase test + + Oxidase test + + Key -/S= Gram negative short rods, A= acid production, no gas production was observed

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Table.3 Reference table for interpretation of result TSI (Bergey s Manual, 9th edition; web link)

REACTION EXPLANATION Acid butt(yellow), alkaline slant (red) Glucose fermented Acid throughout medium, butt and slant yellow Lactose or sucrose or both fermented Gas bubbles in butt, medium sometimes split Aerogenic culture Blackening in the butt Hydrogen Sulfide produced Alkaline slant and butt (medium entirely red) None of the three sugars fermented

Table.4 TSI result

Isolate number Slant Butt Gas Result 1 Yellow Yellow No gas Lactose or sucrose or both production fermented 2 Red Yellow No gas Glucose fermented production Control Yellow Yellow Gas production Lactose or sucrose or both (E.coli) fermented and Aerogenic culture.

Table.5 Effect of chromium on growth of C2

Time (mins) CFU/ml 10 ³ 10 10 Lawn growth 56 20 Lawn growth 17 30 Lawn Growth 15 40 - - 50 - - 60 - -

Photo.1 Glowing Bioluminescent bacterial isolated colonies of C1 and C2 on LA

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Graph.1 Grwoth curve of Isolate C1

Graph.2 Growth curve of Isolate C2

Taken efforts were finally lead to successful pollutant toxicity testing (Engebrech et al., identification of bioluminescent bacteria; 1983; Thomulka and Peck, 1995; Kuruvet et two well isolated colonies of bioluminescent al., 2005; Charrier et al., 2010; Kumar, bacteria from gut flora of Indian Mackerel 2010, Gokhale et al., 2012). This study on luminous agar were identified as Vibrio helped to perform the TVC of the isolate C2 (Bergey s Manual, 9th edition). Growth on TCBS agar confirmed that the bioluminescent isolate belongs to the genus at specific concentration of 10 ³ and 10 in Vibrio. Growth on TCBS agar also state that one of the isolate C2 may be a late sucrose fermenter as we know there are some presence of pollutant chromium of species that shows slow growth and also concentration 1ppm (APHA, 2012; ferment sucrose slowly as they showed late Tchobanoglous, 2004). Chromium is green coloured colonies(Musa et al., 2008; considered nonessential for plants, but an Ransanga et al., 2012). The growth curve of essential trace element in animals. the isolates gave the idea about the log phase Hexavalent compounds have been shown of the organism. In this phase the carcinogenic by inhalation and are corrosive bioluminescent intensity is prominent to tissue. The chromium guidelines for (Engebrech et al., 1983; Kumar, 2010, natural water are linked to the hardness or Gokale et al., 2012). As mentioned above alkalinity of the water (i.e., the softer the isolate C2 showed steady growth in log water, the lower the permitted level for phase i.e. steady intensity of light; so we chromium). The United Nations Food and prefer isolate C2 for further examination in Agriculture Organization recommended

Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 23-32 maximum level for irrigation waters in Berleman, J. 2009. Analysis of inter-species 100 g/L (APHA, 2012; Tchobanoglous, interaction and metabolite excretion in 2004). This minute amount also affects the bioluminescent marine bacteria isolates. bacterial growth as the results showed MBC Microbial diversity. MA. declining number of colonies of organisms; University of Iowa, IA. we may say that the toxicity testing of Boynton, L. 2009. Using bioluminescent chromium in wastewater by bioluminescent bacteria to detect water contaminants. bacteria would be easy and risk free J. U.S. SJWP, 4: 29 41. method(Thomulka and Peck, 1995; Lapota Cao, J.G., Meighen, E.A. 1989. Purification et al., 1998; Boynton. 2009; Charrier et al., and structural identification of an Autoinducer for the luminescence 2010; Ranjith and Karthy, 2011). By this system of vibrio harveyi. J. Bio. Chem., method we can also find out the 264(36): 21670 21676. concentration of the chromium in Charrier, T., Durand, M.J., Affi, M., wastewater or other pollutants like metal Jouanneau, S., Gezekel, H., Thouand, salts zinc, lead acetate, ferrous sulphate etc. G. 2010. Bacterial bioluminescent Modern technique including PCR can help biosensor characterization for on-line to find out the identification of the isolates monitoring of heavy metals pollutions up to species level. in waste water treatment plant effluents. Intech J., Pp. 180 205. The victorious identification of both the Eberhard, A. 1972. Inhibition and activation bioluminescent isolates C1 and C2 to genus of bacterial luciferase synthesis. J. Vibrio states that the method for isolation Bacteriol., 109(3): 1101 1105. and identification was found to be more Engebrech, J., Nealson, K., Silvarman, M. efficient and cost effective, quite easy and 1983. Bacterial bioluminescence: simple. Though the method used for the isolation and genetic analysis from toxicity testing was little bit tricky but Vibrio fischer. Cell, 32: 773 781. helpful to get perfect results and proves the Gokhale, T., Wali, A., Parikh, S., Sood, N. use of bioluminescent bacteria in toxicity 2012. Research of marine isolates in testing is very much simple and harmless to development of biosensor for the performer as well as the environment. environmental pollutants. Eng. Rev., The successful finding suggests that 32(1): 17 22. simplicity and beauty lies within the nature http://cibt.bio.cornell.edu/workshops_and_su itself. mmer_programs/0610ccc/media.pdf http://himedialabs.com/TD/M592.pdf http://microbiologyme.blogspot.in/2013/01/ho References w-to-isolate-glowing-bacteria.html http://www.biology.pl/bakterie_sw/bac_pod_e APHA, 2012. Greenberg, A.E., Clesceri, L.S., n.html Eaton, A.D. 2012. Oxidation of trivalent http://www.scharlabmagyarorszag.hu/katalog chromium: Standard methods for us/01-192_TDS_EN.pdf examination of water and wastewater, https://www.sigmaaldrich.com/content/dam/si 22nd edn. American Public Health gmaaldrich/docs/Sigma/Datasheet/4/p14 Association, American Waste Work 77dat.pdf Association, Water Environment Kannahi, M., Sivshankari, 2014. Isolation and Federation, Part-1000. identification of bioluminescent bacteria Bergey s manual of determinative from marine water at Nagapattanam sea bacteriology, 9th edn, Pp. 190 192. shore. Int. J. Pharm. Sci. Rev., 26(2): 346 351. 32 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 23-32

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